Cancer treatments

ABSTRACT

The invention relates to a product comprised of specific combinations of cell lines intended for use as an allogeneic immunotherapy agent for the treatment of prostate cancer in humans. The heterogeneity of the immunotherapeutic matches the heterogeneity of the antigenic profile in the target prostate cancer and immunises the recipients with many of the potential TAA and TSA which are expressed at various stages of the disease. The invention discloses a vaccine comprising a combination of three different cell lines prepared from primary or metastatic prostate cancer biopsy material. The cell lines are lethally irradiated utilising gamma irradiation at 50-300 Gy to ensure that they are replication incompetent.

This application is a 371 of PCT/GB99/04135 filed on Dec. 9, 1999, whichis hereby incorporated by reference.

FIELD OF THE INVENTION

This invention is concerned with agents for the treatment of primary,metastatic and residual cancer in mammals (including humans) by inducingthe immune system of the mammal or human afflicted with cancer to mountan attack against the tumour lesion. In particular, the inventionpertains to the use of whole-cells, derivatives and portions thereofwith or without vaccine adjuvants and/or other accessory factors. Moreparticularly, this disclosure describes the use of particularcombinations of whole-cells and derivatives and portions thereof thatform the basis of treatment strategy.

BACKGROUND TO THE INVENTION

It is known in the field that cancerous cells contain numerousmutations, qualitative and quantitative, spatial and temporal, relativeto their normal, non-cancerous counterparts and that at certain periodsduring tumour cells' growth and spread a proportion of these are capableof being recognised by the hosts' immune system as abnormal. This hasled to numerous research efforts world-wide to develop immunotherapiesthat harness the power of the hosts' immune system and direct it toattack the cancerous cells, thereby eliminating such aberrant cells atleast to a level that is not life-threatening (reviewed in Maraveyas, A.& Dalgleish, A. G. 1977 Active immunotherapy for solid tumours invaccine design in The Role of Cytokine Networks, Ed. Gregoriadis et al.,Plenum Press, New York, pages 129-145; Morton, D. L. and Ravindranath,M. H. 1996 Current concepts concerning melanoma vaccines in TumorImmunology—Immunotherapy and Cancer Vaccines, ed. Dalgleish, A. G. andBrowning, M., Cambridge University Press, pages 241-268. See also otherpapers in these publications for further detail).

Numerous approaches have been taken in the quest for cancerimmunotherapies, and these can be classified under five categories:

Non-specific Immunotherapy

Efforts to stimulate the immune system non-specifically date back over acentury to the pioneering work of William Coley (Coley, W. B., 1894Treatment of inoperable malignant tumours with toxins of erisipelas andthe Bacillus prodigosus. Trans. Am. Surg. Assoc. 12: 183). Althoughsuccessful in a limited number of cases (e g. BCG for the treatment ofurinary bladder cancer, IL-2 for the treatment of melanoma and renalcancer) it is widely acknowledged that non-specific immunomodulation isunlikely to prove sufficient to treat the majority of cancers. Whilstnon-specific immune-stimulants may lead to a general enhanced state ofimmune responsiveness, they lack the targeting capability and alsosubtlety to deal with tumour lesions which have many mechanisms andplasticity to evade, resist and subvert immune-surveillance.

Antibodies and Monoclonal Antibodies

Passive immunotherapy in the form of antibodies, and particularlymonoclonal antibodies, has been the subject of considerable research anddevelopment as anti-cancer agents. Originally hailed as the magic bulletbecause of their exquisite specificity, monoclonal antibodies havefailed to live up to their expectation in the field of cancerimmunotherapy for a number of reasons including immune responses to theantibodies themselves (thereby abrogating their activity) and inabilityof the antibody to access the lesion through the blood vessels. To date,three products have been registered as pharmaceuticals for human use,namely Panorex (Glaxo-Welicome), Rituxan (IDEC/Genentech/Hoffman laRoche) and Herceptin (Genentech/Hoffman la Roche) with over 50 otherprojects in the research and development pipeline. Antibodies may alsobe employed in active immunotherapy utilising anti-idiotype antibodieswhich appear to mimic (in an immunological sense) cancer antigens.Although elegant in concept, the utility of antibody-based approachesmay ultimately prove limited by the phenomenon of ‘immunological escape’where a subset of cancer cells in a mammalian or human subject mutatesand loses the antigen recognised by the particular antibody and therebycan lead to the outgrowth of a population of cancer cells that are nolonger treatable with that antibody.

Subunit Vaccines

Drawing on the experience in vaccines for infectious diseases and otherfields, many researchers have sought to identify antigens that areexclusively or preferentially associated with cancer cells, namelytumour specific antigens (TSA) or tumour associated antigens (TAA), andto use such antigens or fractions thereof as the basis for specificactive immunotherapy.

There are numerous ways to identify proteins or peptides derivedtherefrom which fall into the category of TAA or TSA. For example, it ispossible to utilise differential display techniques whereby RNAexpression is compared between tumour tissue and adjacent normal tissueto identify RNAs which are exclusively or preferentially expressed inthe lesion. Sequencing of the RNA has identified several TAA and TSAwhich are expressed in that specific tissue at that specific time, buttherein lies the potential deficiency of the approach in thatidentification of the TAA or TSA represents only a “snapshot” of thelesion at any given time which may not provide an adequate reflection ofthe antigenic profile in the lesion over time. Similarly a combinationof cytotoxic T lymphocyte (CTL) cloning and expression-cloning of cDNAfrom tumour tissue has lead to identification of many TAA and TSA,particularly in melanoma. The approach suffers from the same inherentweakness as differential display techniques in that identification ofonly one TAA or TSA may not provide an appropriate representation of aclinically relevant antigenic profile.

Over fifty such subunit vaccine approaches are in development for thetreatment of a wide range of cancers, although none has yet receivedmarketing authorisation for use as a human pharmaceutical product. In asimilar manner to that described for antibody-based approaches above,subunit vaccines may also be limited by the phenomenon of immunologicalescape.

Gene Therapy

The majority of gene therapy trials in human subjects have been in thearea of cancer treatment, and of these a substantial proportion havebeen designed to trigger and/or amplify patients' immune responses. Ofparticular note in commercial development are Allovectin-7 andLeuvectin, being developed by Vical Inc for a range of human tumours,CN706 being developed by Calydon Inc for the treatment of prostatecancer, and StressGen Inc.'s stress protein gene therapy for melanomaand lung cancer. At the present time, it is too early to judge whetherthese and the many other ‘immuno-gene therapies’ in development bycommercial and academic bodies will ultimately prove successful, but itis widely accepted that commercial utility of these approaches arelikely to be more than a decade away.

Cell-based Vaccines

Tumours have the remarkable ability to counteract the immune system in avariety of ways including: downregulation of the expression of potentialtarget proteins; mutation of potential target proteins; downregulationof surface expression of receptors and other proteins; downregulation ofMHC class I and II expression thereby disallowing direct presentation ofTAA or TSA peptides; downregulation of co-stimulatory molecules leadingto incomplete stimulation of T-cells leading to anergy; shedding ofselective, non representative membrane portions to act as decoy to theimmune system; shedding of selective membrane portions to anergise theimmune system; secretion of inhibitory molecules; induction of T-celldeath; and many other ways. What is clear is that the immunologicalheterogeneity and plasticity of tumours in the body will have to bematched to a degree by immunotherapeutic strategies which similarlyembody heterogeneity. The use of whole cancer cells, or crudederivatives thereof, as cancer immunotherapies can be viewed asanalogous to the use of whole inactivated or attenuated viruses asvaccines against viral disease. The potential advantages are:

(a) whole cells contain a broad range of antigens, providing anantigenic profile of sufficient heterogeneity to match that of thelesions as described above;

(b) being multivalent (i.e. containing multiple antigens), the risk ofimmunological escape is reduced (the probability of cancer cells‘losing’ all of these antigens is remote); and

(c) cell-based vaccines include TSAs and TAAs that have yet to beidentified as such; it is possible if not likely that currentlyunidentified antigens may be clinically more relevant than therelatively small number of TSAs/TAAs that are known.

Cell-based vaccines fall into two categories. The first, based onautologous cells, involves the removal of a biopsy from a patient,cultivating tumour cells in vitro, modifying the cells throughtransfection and/or other means, irradiating the cells to render themreplication-incompetent and then injecting the cells back into the samepatient as a vaccine. Although this approach enjoyed considerableattention over the past decade, it has been increasingly apparent thatthis individually-tailored therapy is inherently impractical for severalreasons. The approach is time consuming (often the lead time forproducing clinical doses of vaccine exceeds the patients' lifeexpectancy), expensive and, as a ‘bespoke’ product, it is not possibleto specify a standardised product (only the procedure, not the product,can be standardised and hence optimised and quality controlled).Furthermore, the tumour biopsy used to prepare the autologous vaccinewill have certain growth characteristics, interactions and communicationwith surrounding tissue that makes it somewhat unique. This alludes to apotentially significant disadvantage to the use of autologous cells forimmunotherapy: a biopsy which provides the initial cells represents animmunological snapshot of the tumour, in that environment, at that pointin time, and this may be inadequate as an immunological representationover time for the purpose of a vaccine with sustained activity that canbe given over the entire course of the disease.

The second type of cell-based vaccine and the subject of the currentinvention describes the use of allogeneic cells which are be genetically(and hence immunologically) mismatched to the patients. Allogeneic cellsbenefit from the same advantages of multivalency as autologous cells. Inaddition, as allogeneic cell vaccines can be based on immortalised celllines which can be cultivated indefinitely in vitro, thus this approachdoes not suffer the lead-time and cost disadvantages of autologousapproaches. Similarly the allogeneic approach offers the opportunity touse combinations of cells types which may match the disease profile ofan individual in terms of stage of the disease, the location of thelesion and potential resistance to other therapies.

There are numerous published reports of the utility of cell-based cancervaccines (see, for example, Dranoff, G. et al. WO 93/06867; Gansbacher,P. WO 94/18995; Jaffee, E. M. et al. WO 97/24132; Mitchell, M. S. WO90/03183; Morton, D. M. et al. WO 91/06866). These studies encompass arange of variations from the base procedure of using cancer cells as animmunotherapy antigen, to transfecting the cells to produce GM-CSF,IL-2, interferons or other immunologically-active molecules and the useof ‘suicide’ genes. Groups have used allogeneic cell lines that areHLA-matched or partially-matched to the patients' haplotype and alsoallogeneic cell lines that are mismatched to the patients' haplotype inthe field of melanoma and also mismatched allogeneic prostate cell linestransfected with GM-CSF.

DESCRIPTION OF THE INVENTION

The invention disclosed here relates to a product comprised of specificcombinations of cell lines intended for use as an allogeneicimmunotherapy agent for the treatment of prostate cancer in humans. Theheterogeneity of the immunotherapeutic described herein matches theheterogeneity of the antigenic profile in the target prostate cancer andimmunises the recipients with many of the potential TAA and TSA whichare expressed at various stages of the disease. The cell lines arechosen from appropriate cell lines which possess the followingcharacteristics: the cells are immortalised, prostate or metastaticprostate in origin, show good growth in large scale cell culture, andare well characterised allowing for quality control and reproducibleproduction of the component cell lines.

The invention disclosed herein also relates to a product comprising of acombination of cells lines described above whereby the cell lines arechosen to allow for the maximum mismatch of haplotype with the intendedpatient population, thereby ensuring the maximum allogeneic potentialand subsequent immune response to the product.

The invention described discloses a vaccine comprising a combination ofthree different cell lines prepared from primary or metastatic prostatecancer biopsy material using methods known in the art (reviewed andcited in Rhim, J. S. and Kung, H-F., 1997 Critical Reviews inOncogenesis 8(4):305-328) and/or selected from Group A (cell linesderived from primary prostate cancer lesions) and Group B (cell linesderived from metastatic prostate cancer lesions) listed in Table 1.

In one embodiment, the combination of cell lines consists of threedifferent cell lines derived from primary prostate cancer lesions.

In another embodiment, the combination consists of two different celllines derived from primary prostate cancer lesions and one cell linederived from a metastatic prostate cancer lesion.

In another embodiment, the combination consists of one cell line derivedfrom a primary prostate cancer lesion combined with two different celllines derived from metastatic prostate cancer lesions.

In a further embodiment, the combination consists of three differentcell lines derived from metastatic prostate cancer lesions.

The cell lines are lethally irradiated utilising gamma irradiation at50-300 Gy to ensure that they are replication incompetent.

The cell lines and combinations referenced above, to be useful asimmunotherapy agents must be frozen to allow transportation and storage,therefore a further aspect of the invention is any combination of cellsreferenced above formulated with a cryoprotectant solution. Suitablecryoprotectant solutions may include but are not limited to, 10-30% v/vaqueous glycerol solution, 5-20% v/v dimethyl sulphoxide or 5-20% w/vhuman serum albumin may be used either as single cryoprotectants or incombination.

TABLE 1 Group A Group B NIH1519-CPTX, NIH1532-CP2TX, DU145 (ATCC Number:NIH1535-CP1TX and NIH1542-CP3TX HTB-81) (immortalised lines derived fromLnCap (ATCC Number: primary prostate cancers by Dr. CRL-1740 andCRL-10995) Suzanne Topalian at the NIH; PC3 (ATCC Number: these celllines have been CRL-1435) described in Cancer Research, vol 57 (5), pp995-1002 and have been deposited at ATCC for patent purposes) CA-HPV-10(ATCC Number: CRL-2220)

A further embodiment of the invention is the use of the cell linecombinations with non-specific immune stimulants such as BCG or M.Vaccae, Tetanus toxoid, Diphtheria toxoid, Bordetella Pertussis,interleukin 2, intedeukin 12, interleukin 4, interleukin 7, CompleteFreund's Adjuvant, Incomplete Freund's Adjuvant or other non-specificagents known in the art. The advantage is that the general immunestimulants create a generally enhanced immune status whilst thecombinations of cell lines, both add to the immune enhancement throughtheir haplotype mismatch and target the immune response to a plethora ofTAA and TSA as a result of the heterogeneity of their specific origins.

The invention will now be described with reference to the followingexamples, and the Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows T-cell proliferation data for Patient Nos. 202 and 205;

FIG. 2 shows Western Blot analysis of serum from Patient Nos. 201 and203;

FIG. 3 shows Antibody Titres of serum from Patient No. 201; and

FIG. 4 shows PSA data for Patients 201 and 208.

EXAMPLE 1 Growth, Irradiation, Formulation and Storage of Cells

An immortalised cell line derived from primary prostate tissue, namelyNIH1542-CP3TX, was grown in roller bottle culture in KSFM mediumsupplemented with 25 μg/ml bovine pituitary extract, 5 ng/ml ofepidermal growth factor, 2 mM L-glutamine, 10 mM HEPES buffer and 5%foetal calf serum (FCS) (hereinafter called “modified KSFM”) followingrecovery from liquid nitrogen stocks. Following expansion in T175 staticflasks the cells were seeded into roller bottles with a growth surfacearea of 1,700 cm² at 2-5×10⁷ cells per roller bottle.

Two metastasis-derived cell lines were also used, namely LnCap and Du145both of which were sourced from ATCC. LnCap was grown in large surfacearea static flasks in RPMI medium supplemented with 10% FCS and 2 mML-glutamine following seeding at 1-10×10⁶ cells per vessel and thengrown to near confluence. Du145 was expanded from frozen stocks instatic flasks and then seeded into 850 cm² roller bottles at 1-20×10⁷cells per bottle and grown to confluence in DMEM medium containing 10%FCS and 2 mM L-glutamine.

All cell lines were harvested utilising trypsin at 1× normalconcentration. Following extensive washing in DMEM the cells werere-suspended at a concentration of 10—40×10⁶ cells/ml and irradiated at50-300 Gy using a Co⁶⁰ source. Following irradiation the cells wereformulated in cryopreservation solution composed of 10% DMSO, 8% humanserum albumin in phosphate buffered saline, and frozen at a cellconcentration of 15-50×10⁶ cells/ml by cooling at a rate of 1° C. perminute and then transferred into a liquid nitrogen freezer untilrequired for use.

Vaccination

Prostate cancer patients were selected on the basis of being refractoryto hormone therapy with a serum PSA level of 30 ng/ml. Ethicalpermission and MCA (UK Medicines Control Agency) authorisation weresought and obtained to conduct this trial in 15 patents.

The vaccination schedule was as follows:

Dose Number Cell Lines Administered 1, 2 and 3 NIH1542-CP3TX (24 × 10⁶cells per dose) 4 and subsequent LnCap/Du145/NIH1542 (8 × 10⁶ cells ofeach cell line per dose)

The cells were warnned gently in a water bath at 37° C. and admixed withmycobacterial adjuvant prior to injection into patients. Injections weremade intra-dermally at four injection sites into draining lymph nodebasins. The minimum interval between doses was two weeks, and most ofthe doses were given at intervals of four weeks. Prior to the firstdose, and prior to some subsequent doses, the patients were tested fordelayed-type hypersensitivity (DTH) against the three cell lines listedin the vaccination schedule above and also against PNT2 (an immortalizednormal prostate epithelial cell line sourced from ECACC) (all testsinvolved 0.8×10⁶ cells with no adjuvant).

Analysis of Immunological Response

(a) T-Cell Proliferation Responses

To determine if vaccination resulted in a specific expansion of T-cellpopulations that recognised antigens derived from the vaccinating celllines we performed a proliferation assay on T-cells followingstimulation with lysates of the prostate cell lines. Whole blood wasextracted at each visit to the clinic and used in a BrdU(bromodeoxyuridine) based proliferation assay as described below:

Patient BrdU Proliferation Method

Reagents RPMI Life Technologies, Paisley Scotland. BrdU Sigma ChemicalCo, Poole, Dorset. PharMlyse 35221E Pharmingen, Oxford UKCytofix/Cytoperm 2090KZ ″ Perm/Wash buffer (×10) 2091KZ ″ FITCAnti-BrdU/Dnase 340649 Becton Dickinson PerCP Anti-CD3 347344 ″ PeAnti-CD4 30155X Pharmingen Pe Anti-CD8 30325X ″ FITC mu-IgG1 349041Becton Dickinson PerCP IgG1 349044 ″ PE IgG1 340013 ″

Method

1) Dilute 1 ml blood with 9 ml RPMI+2mM L-gin +PS +50 μM 2-Me. Do notadd serum. Leave overnight at 37° C.

2) On following morning, aliquot 450 μl of diluted blood into wells of a48-well plate and add 50 μl of stimulator lysate. The lysate is made byfreeze-thawing tumour cells (2×10⁶ cell equivalents/ml)×3 in liquidnitrogen and then storing aliquots frozen until required.

3) Culture cells at 37° C. for 5 days

4) On the evening of day 5 add 50 μl BrdU@30 μg/ml

5) Aliquot 100 μl of each sample into a 96-well round-bottomed plate.

6) Spin plate and discard supernatant

7) Lyse red cells using 100 μl Pharmlyse for 5 minutes at roomtemperature

8) Wash ×2 with 50 μl of Cytofix

9) Spin and remove supernatant by flicking

10) Permeabilise with 100 μl Perm wash for 10 mins at RT

11) Add 30 μl of antibody mix comprising antibodies at correct dilutionmade up to volume with Perm-wash

12) Incubate for 30 mins in the dark at room temperature.

13) Wash ×1 and resuspend in 100 μl 2% paraformaldehyde

14) Add this to 400 μl FACSFlow in cluster tubes ready for analysis

15) Analyse on FACScan, storing 3000 gated CD3 events.

96-well plate for stimulation

Nil ConA 1542 LnCap Du145 Pnt2 PBL 1 PBL 2 PBL 3 PBL 4 PBL 5 PBL 6

PBL 1 PBL 2 PBL 3 PBL 4 PBL 5 PBL 6 Nil A 15 D Nil A 15 D Nil A 15 D NilA 15 D Nil A 15 D Nil A 15 D Nil D 15 E Nil D 15 E Nil D 15 E Nil D 15 ENil D 15 E Nil D 15 E Nil E Ln D Nil E Ln D Nil E Ln D Nil E Ln D Nil ELn D Nil E Ln D Con D Ln E Con D Ln E Con D Ln E Con D Ln E Con D Ln ECon D Ln E Con E Du D Con E Du D Con E Du D Con E Du D Con E Du D Con EDu D Du E Du E Du E Du E Du E Du E Pn D Pn D Pn D Pn D Pn D Pn D Pn E PnE Pn E Pn E Pn E Pn E Legend: A: IgG1-FITC (5 μl) IgG1-PE (5 μl)IgG1-PerCP (5 μl) 15 μl MoAb + 15 μl D: BrdU-FITC (5 μl) CD4-PE (5 μl)CD3-PerCP (5 μl) 15 μl MoAB + 15 μl E: BrdU-FITC (5 μl) CD8-PE (5 μl)CD3-PerCP (5 μl) 15 μl MoAb + 15 μl 15: NIH1542-CP3TX Ln: LnCap D: Du145Pn: PNT2 Con: ConA lectin (positive control) Nil No stimulation

The results for the proliferation assays are shown in FIG. 1 where aproliferation index for either CD4 or CD8 positive T-cells are plottedagainst the various cell lysates, the proliferation index being derivedby dividing through the percentage of T-cells proliferating by theno-lysate control.

Results are shown for patient numbers 202 and 205. Results are given forfour cell lysates namely, NIH1542, LnCap, DU-145 and PNT-2 (animmortalised normal prostate epithelial cell line). Overall, 50% ofpatients treated mount a specific proliferative response toNIH1542-CP3TX, LnCap and DU-145 to a degree and in some cases also toPNT-2.

(b) Western Blots Utilising Patients' Serum

Standardised cell lysates were prepared for a number of prostate celllines to enable similar quantites of protein to be loaded on adenaturing SDS PAGE gel for Western blot analysis. Each blot was loadedwith molecular weight markers, and equal amounts of protein derived fromcell lysates of NIH1542, LnCap, DU-145 and PNT-2. The blot was thenprobed with serum from patients derived from pre-vaccination andfollowing 16 weeks vaccination (four to six doses).

Method

a) Sample Preparation (Prostate Tumor Lines)

Wash cell pellets 3 times in PBS

Re-suspend at 1×10⁷ cells/ml of lysis buffer

Pass through 5 cycles of rapid freeze thaw lysis in liquidnitrogen/water bath

Centrifuge at 1500 rpm for 5 min to remove cell debris

Ultracentrifuge at 20,000 rpm for 30 min to remove membrane contaminants

Aliquot at 200 μl and stored at −80° C.

b) Gel Electrophoresis

Lysates mixed 1:1 with Laemelli sample buffer and boiled for 5 min

20 μg samples loaded into 4-20% gradient gel wells

Gels run in Bjerrum and Schafer-Nielson transfer buffer (with SDS) at200 V for 35 min.

c) Westem Transfer

Gels, nitrocellulose membranes and blotting paper equilibrated intransfer buffer for 15 min

Arrange gel-nitrocellulose sandwich on anode of semi-dry electrophoretictransfer cell: 2 sheets of blotting paper, nitrocellulose membrane, gel,2 sheets of blotting paper

Apply cathode and run at 25 V for 90 min.

d) Immunological Detection of Proteins

Block nitrocellulose membranes ovenight at 4° C. with 5% Marvel inPBS/0/05% Tween 20

Rinse membranes twice in PBS/0.05% Tween 20, then wash for 20 min and2×5 min at RT on a shaking platform

Incubate membranes in 1:20 dilution of clarified patient plasma for 120min at RT on a shaking platform

Wash as above with an additional 5 min final wash

Incubate membranes in 1:250 dilution of biotin anti-human IgG or IgM for90 min at RT on a shaking platform

Wash as above with an additional 5 min final wash

Incubate membranes in 1:1000 dilution of streptavidin-horseradishperoxidase conjugate for 60 min at RT on a shaking platform

Wash as above

Incubate membranes in Diaminobenzidine peroxidase substrate for 5 min toallow colour development, stop reaction by rinsing membrane with water

Results of Western blots probed with anti-IgG second antibodies forpatients 201 and 203 are shown in FIG. 2. The Figure shows baseline andweek 16 time points for each patient with four cell lysates on eachblot.

Overall in patients who received at least four to six doses, over 50%showed an increase in intensity of bands present before vaccinationand/or a broadening of the number of bands being recognised by theserum.

Of particular note is the reactivity of serum from patients 201 and 203towards the PNT2 lysate which did not form part of the vaccinationregime (other than DTH testing), but nevertheless appears to sharecommon antigens with NIH1542, LnCap and DU145 in both patients serum.

(c) Antibody Titre Determination

Antibody titres were determined by coating ELISA plates withstandardised cell line lysates and performing dilution studies on serumfrom patients vaccinated with the cell lines.

Method for ELISA with anti-lysate IgG.

1. Coat plates with 50 μl/well lysates (@10 μg/ml) using the followingdilutions:

Lysate Protein conc Coating conc amount/ml amount in 5 mls μl PNT2 2.5mg/ml 10 μg/ml 3.89 μl 19.4 μl 1542 4.8 mg/ml 10 μg/ml 2.07 μl 10.3 μlDu145 2.4 mg/ml 10 μg/ml 4.17 μl 20.8 μl LnCap 2.4 mg/ml 10 μg/ml 4.12μl 20.6 μl

2. Cover and incubate overnight@4° C.

3. Wash ×2 PBS-Tween. Pound plate on paper towels to dry.

4. Block with PBS/10% FCS (100 μl/well)

5. Cover and incubate@room temperature for 1 hour (minimum).

6. Wash ×2 PBS-Tween

7. Add 100 μl PBS-10% FCS to rows 2-8

8. Add 200 μl plasma sample (diluted 1 in 100 in PBS-10% FCS ie. 10 μlplasma added to 990 μls PBS- 10% FCS) to row 1 and do serial 100 μldilutions down the plate as below. Discard extra 100 μl from bottomwell. Cover and incubate in fridge overnight.

9. Dilute biotinylated antibody (Pharmingen; IgG 34162D) ie. final conc1 mg/ml (ie 20 ml in 10 mls).

10. Cover and incubate@RT for 45min.

11. Wash ×6 as above.

12. Dilute streptavidin —HRP (Pharmingen, 13047E 0; dilute 1:1000 (ie 10ml →10 mls).

13. Add 100 ml/well.

14. Incubate 30 min@RT.

15. Wash ×8.

16. Add 100 ml substrate/well. Allow to develop 10-80 min at RT.

17. Colour reaction stopped by adding 100 ml 1M H2SO4.

18. Read OD@A405nm.

The results (FIG. 3) show that after vaccination with at least four tosix doses, patients can show an increase in antibody titre against cellline lysates.

(d) Evaluation of PSA Levels

PSA levels for patients receiving the vaccine were recorded at entryinto the trial and throughout the course of vaccination, using routinelyused clinical kits. The PSA values for patients 201 and 208 are shown inFIG. 4 and portray a drop or stabilisation of the PSA values, which inthis group of patients usually continues to rise, often exponentially.The result for patient 201 is somewhat confounded by the radiotherapytreatment to alleviate bone pain, although the PSA level had droppedsignificantly prior to radiotherapy.

EXAMPLE 2

The invention can also be applied to earlier stage prostate cancerpatients, and the immunotherapy can also be administered throughdifferent routes. As an example, the following protocol can be used:

Cells are grown, irradiated, formulated and stored according to themethods described in Example 1. Prostate cancer patients are selectedprior to radical prostatectomy and are vaccinated with a combination ofthree irradiated cell lines (8×10⁶ cells per line) three times at twoweek intervals prior to surgery. Approximately half of the patients arevaccinated intradermally into four draining lymph node basins (celllines mixed with mycobacterial adjuvant for at least the first dose);remaining patients are injected intra-prostatically, with intradermalmycobacterial adjuvant administered at a distant site for at least thefirst dose. Biopsy samples of the prostate removed by surgery areexamined for prostate cell death and the presence of infiltrating immunecells. In addition, T-cell function, Western blot analysis and antibodytitres are determined according to the method of Example 1. Serum PSA isalso measured at intervals in these patients.

Following this protocol, immunological responses can be detected. Inaddition, death of prostate cells can be detected in surgical biopsies.

What is claimed is:
 1. An allogeneic immunotherapeutic agent for thetreatment of prostate cancer comprising three human prostate tumor celllines of which one cell line is derived from a primary tumor and theother two cell lines are derived from metastatic tissue.
 2. Anallogeneic immunotherapeutic agent for the treatment of prostate cancercomprising a mixture of three human prostate tumor cell lines, whereinone cell line is derived from a primary tumor and the other two celllines are derived from two different metastatic tissues.
 3. Anallogeneic immunotherapeutic agent for the treatment of prostate cancercomprising a mixture of three human prostate tumor cell lines, whereinthree cell lines are derived from three different primary tumors.
 4. Anallogeneic immunotherapeutic agent for the treatment of prostate cancercomprising a mixture of three human prostate tumor cell lines, whereintwo cell lines are derived from one or two primary tumor(s) and theother cell line is derived from a metastatic tissue.
 5. An allogeneicimmunotherapeutic agent for the treatment of prostate cancer comprisinga mixture of three human prostate tumor cell lines, wherein three celllines are derived from metastatic tissues.
 6. An allogeneicimmunotherapeutic agent for the treatment of prostate cancer comprisinga mixture of three human prostate tumor cell lines, wherein three celllines are derived from two or three different metastatic tissues.
 7. Animmunotherapeutic agent of claim 1, wherein the tumor cell lines derivedfrom metastatic tissue and are selected from the group consisting ofLnCap, DU145 and PC3.
 8. An immunotherapeutic agent of claim 1, whereinthe tumor cell lines have been irradiated at 50 to 300 Gy.
 9. Animmunotherapeutic agent of claim 1, wherein the tumor cell lines havebeen irradiated at 100 to 150 Gy.
 10. An allogeneic immunogeniccomposition comprising an immunotherapeutic agent of claim 1 combinedwith a vaccine adjuvant selected from the group consisting of BCG, M.Vaccae, Tetanus toxoid, Diphtheria toxoid, Bordetella Pertussis,interteukin 2, interleukin 12, interleukin 4, interleukin 7, CompleteFreund's Adjuvant, Incomplete Freund's Adjuvant, and a nonspecificadjuvant.
 11. An immunogenic composition comprising an immunotherapeuticagent of claim 1 combined with a vaccine adjuvant, wherein the adjuvantis a mycobacterial preparation.
 12. An immunotherapeutic agent of claim1, wherein the cells are formulated with a cryoprotectant solutionincluding at least one selected from the group consisting of 10-30% v/vaqueous glycerol solution, 5-20% v/v dimethyl sulphoxide and 5-20% w/vhuman serum albumin.
 13. An immunotherapeutic agent of claim 1, whereinthe cells are formulated with a cryoprotectant solution comprising 5-20%v/v dimethyl sulphoxide and 5-20% w/v human serum albumin incombination.
 14. An immunotherapeutic agent of claim 1, wherein saidagent is capable of inducing an immune response in patients byactivation of immune T-cells.
 15. An immunotherapeutic agent of claim 1,wherein said agent is capable of inducing an immune response in patientsby induction of antibody production.
 16. An immunotherapeutic agent ofclaim 1, wherein said agent is capable of inducing a decrease in therate of rise or a decline in the level of serum PSA in prostate cancerpatients.
 17. An immunotherapeutic agent according to claim 1, whereinsaid agent is capable of being administered intradermally.
 18. Animmunotherapeutic agent according to claim 1, wherein said agent iscapable of being administered intra-prostatically.
 19. An allogeneicimmunotherapeutic vaccine composition for the treatment of prostatecancer, wherein said composition comprises an agent according to claim 1and a physiologically acceptable agent selected from the groupconsisting of excipient, adjuvant and carrier.
 20. An allogeneic methodof prophylaxis or treatment of prostate cancer by administering to apatient an effective amount of an agent according to claim
 1. 21. Anallogeneic immunotherapeutic agent for the treatment of prostate cancercomprising three different human prostate tumor cell lines, wherein onecell line is derived from a primary prostate tumor and the other twocell lines are derived from metastatic prostate tissue.
 22. Theallogeneic immunotherapeutic agent of claim 21, where the cell lines areselected so as to maximize haplotype mismatch.
 23. An allogeneicimmunotherapeutic agent for the treatment of prostate cancer comprisingthree different human prostate tumor cell lines, wherein one cell lineis derived from a metastatic prostate tissue and the other two celllines are derived from primary prostate turmors.
 24. The allogeneicimmunotherapeutic agent of claim 23, where the cell lines are selectedso as to maximize haplotype mismatch.